The present invention relates to methods of identifying a patient who has an altered immune status compared to a normal status. The methods involve determining an immune status index for the patient and comparing the value of the index to the immune status index in healthy individuals. A significant variation between the patient's immune status index and the immune status index for healthy individuals indicates that the patient's immune status is altered. The present invention is used to identify patients with immunosuppression, hypersensitivity or autoimmunity as well as to monitor the immune response in general to facilitate medical treatment. The immune status index is used to stage or evaluate the progress of cancer therapy including chemotherapy, immunotherapy or surgery. The immune status index is used to evaluate a patient undergoing organ transplant and to evaluate the effect of ongoing therapy for autoimmune diseases or allergies.
The immune system is comprised of a complex array of precisely regulated cell types and the soluble molecules which these cells secrete. The immune response in a healthy individual involves recognition of a pathogen, other foreign material, or tumor cell followed by the elimination of the pathogen or other foreign material from the organism. Broadly speaking, the immune response can be divided into two categories, the innate responses and the adoptive responses. As a result of interactions among the components of the immune system, however, most immune responses comprise a variety of innate and adoptive mechanisms.
The innate responses are generally mediated by an important group of leukocytes known as phagocytic cells which include monocytes, macrophages and polymorphonuclear neutrophils. In general, these cell types act as a first line of defense against infection because they utilize non-specific recognition systems to bind microorganisms, internalize them and destroy them.
Central to the adoptive responses of the immune system are the lymphocytes. Lymphocytes specifically recognize individual pathogens whether they are inside host cells or outside cells in blood or in tissue fluids. Lymphocytes are generally divided into two groups, T lymphocytes (also called T cells) and B lymphocytes (also called B cells). The B cells release specific antibodies that combat extracellular pathogens and their products by binding to specific target molecules. T cells, on the other hand, have a wider array of responsibilities. Certain T cells interact with phagocytic cells to help the phagocytes destroy pathogens they have taken up. Other T cells recognize aberrant cells or cells infected by virus and destroy them. Still other T cells control B cell development and antibody production.
A definitive T cell marker is the T cell antigen receptor designated TCR. Among T cells in the blood, generally more than 95% of them are classified as TCR-2 and the remainder are TCR-1. TCR-1 and TCR-2 are distinguished on the basis of Ti subunits. The Ti subunits of the TCR-2 are two disulfide-linked polypeptides known as .alpha. and .beta.. TCR-1 is structurally similar to TCR-2, but the TCR-1 Ti subunits are the .gamma. and .delta. polypeptides. Both TCR-1 and TCR-2 are associated with a complex of polypeptides which comprise the CD3 complex.
The TCR found on the surface of all T cells is composed of at least six different subunits which can be divided into three distinct subgroups of proteins. Klausner (1990). The heterodimers .alpha..beta. or .gamma..delta. within the receptor complex are responsible for ligand binding. Another subgroup of proteins which comprise the TCR are the CD3 chains which encompass at least four distinct, but closely related subunits. These subunits are .gamma., .delta., .epsilon. and .zeta.. Koning (1990); Blumberg (1990). Diversification of receptor types is the result of segregation of chains of the TCR complex into multiple subunits. Incompletely assembled complexes are degraded, resulting in the surface expression of only completely assembled receptors. Klausner (1989).
T cells that are TCR-2 are subdivided into a subset of cells which carry the CD4 marker and another which carries the CD8 marker. The CD4.sup.+ subset (TH) mainly induces immune responses while the CD8.sup.+ subset (Tc) is largely composed of cytotoxic/suppressor cells. The CD4.sup.+ subset is subdivided into those cells which positively influence the response of T cells and B cells. Another CD4.sup.+ subset of cells induces the suppressor/cytotoxic functions of CD8.sup.+ cells.
The CD4.sup.+ subset is further subdivided into TH-1 and TH-2 type cells. TH-1 and TH-2 type cells are distinguished on the basis of the spectrum of lymphokines they secrete. TH-1 cells have been found to secrete interleukin-2 (IL-2) and IFN-.gamma., while TH-2 cells have been found to secrete IL-4, IL-5, IL-6 and IL-10. TH-1 and TH-2 cell types are thought to be derived from a common precursor population termed a TH-0 cell. In contrast to the mutually exclusive cytokine production of all or most of TH-1 and TH-2 cells, TH-0 cells produce all or most of these lymphokines. Treatment of TH-0 cells with IL-12 results in the production of TH-1-type cells. IL-12 is produced by macrophages and B cells.
TH cells appear to control and modulate the development of immune responses. TH cells play a major role in determining which epitopes become targets of the immune response and selection of effector mechanisms. The antigen-presenting cells (APCs) present processed antigen-to TH cells which recognize certain epitopes and thus select those which act as targets for the relevant effector functions. The TH cells then select and activate the appropriate effector cells including B cells that produce antibody and modulate the actions of other effector cells, Tc cells, natural killer (NK) cells, macrophages, granulocytes and antibody dependent cytotoxic (K) cells.
The release of different cytokines by TH cells may play a role in selection of effector mechanisms and cytotoxic cells. TH-1 cells secrete IL-2 and IFN-.gamma. which tend to activate macrophages and cytotoxic cells. In contrast, TH-2 cells secrete IL-4, IL-5, IL-6 and IL-10 and tend to increase production of eosinophils and mast cells as well as enhance production of antibody including IgE and decrease the function of cytotoxic cells. Once established, the TH-1 or TH-2 pattern is maintained through production of a cytokine that inhibits production of the other subset. The IFN-.gamma. produced by TH-1 cells inhibits production of TH-2 type cytokines such as IL-4, IL-10 while the IL-10 produced by TH-2 inhibits production of TH-1 type cytokines such as IL-2 and .gamma.IFN.
In addition to determining which epitopes are to be the targets of the immune system, the immune system must also select the appropriate effector mechanisms for each infection. Effector mechanisms which can be selected include 1) cytotoxic T cell, 2) antibody plus mast cells and eosinophils or 3) macrophage activation and delayed hypersensitivity. Activation of inappropriate effector mechanisms can lead to enhanced susceptibility rather than protection.
The molecular mechanism by which T cell clones become restricted to express only certain lymphokine genes has remained obscure, although it has been reported that cAMP, or a labile regulatory protein, can inhibit expression of IL-2 in TH-2 cells. Novak (1990), Munoz (1989). Human B cell lines are capable of producing endogenous .gamma.IFN and this gene expression correlates, at least in part, with the methylation status of a SnaB 1 restriction enzyme site (TACGTA) present between the CAAAT and TATA box in the human .gamma.IFN promoter. Pang (1992). The SnaB 1 enzyme is methylation sensitive as it does not cleave DNA if the C is methylated at the 5 position, but does cleave DNA if the C is not methylated. Yang (1990). In a human B-cell line that expresses .gamma.IFN spontaneously, and in a murine T-cell line stably transfected with the human .gamma.IFN genomic DNA, this site was totally hypomethylated and completely cleaved by SnaB 1. Pang (1992).
Tc cells, also known as killer T cells, are effector cells which play an important role in immune reactions against intracellular parasites and viruses by lysing infected target cells. Cytotoxic T cells have also been implicated in protecting the body from developing cancers through an immune surveillance mechanism. Under certain conditions, CD8.sup.+ T cells have also been shown to function as cells able to suppress the immunologic activity. This is mediated by the production of the raw factors produced by the TH-2 cells; i.e. IL4, IL10. T suppressor cells block the induction and/or activity of T helper cells. T cells do not generally recognize free antigen, but recognize it on the surface of other cells. These other cells may be specialized antigen-presenting cells capable of stimulating T cell division or may be virally-infected cells within the body that become targets for cytotoxic T cells.
Tc/Ts cells usually recognize antigen in association with class I Major Histocompatibility Complex (MHC) products which are expressed on all nucleated cells. Helper T cells, and most T cells which proliferate in response to antigen in vitro, recognize antigen in association with class II MHC products. Class II products are expressed mostly on antigen-presenting cells and on some lymphocytes.
In summary, the process of activation of the humoral (antibody and complement) or the cellular arm of the immune response and the regulation of such response appear to be controlled by the production of cytokines by T-cells and monocytes. Thus, it is likely that alterations in this regulation could result in the abnormal function of the immune response. This abnormal function could either be a decreased immune response resulting in immunosuppression, or alternatively in an abnormally increased response against one's own normal tissues in what is known as autoimmunity.
Determining the status of the immune response has mainly been done by clinical means. An "opportunistic infection," that is, the presence of an infection by a microorganism that normally is not pathogenic, suggests an immunosuppressed state. Alternatively, the presence of rheumatoid arthritis suggests an autoimmune process. Once the clinical findings occur, specific laboratory tests can confirm these findings. These laboratory tests mainly confirm that an altered immune system exists, for example, the antinuclear antibody test demonstrates the presence of autoantibodies in the serum of lupus patients, or the isolation of an opportunistic microorganism confirms the presence of an immunosuppressive process. However, there are no adequate tests to monitor the function of the immune system. Present immune tests on immune function include:
(1) Cell number: White blood cell count, CD4.sup.+ /CD8.sup.+ ratio. PA1 (2) Cell response: Proliferation index to tetanus toxoid. PA1 (3) Antibody levels in serum. PA1 (4) Lymphokine production: Tests absolute levels of lymphokines in serum. PA1 1. a. a kit for cell separation including a column to eliminate B cells, granulocytes and monocytes; PA1 2. a. a kit for cell separation including a column to eliminate B cells, granulocytes and monocytes;
None of these tests take into account the fact that the immune response is a balance between TH-1 and TH-2 responses. Considering the complex number of different specialized cell types that comprise the immune system, as well as the subtle control networks that exist among these cell types, it is not surprising that even small perturbations in this system can lead to serious illness in the patient. Many diseases are characterized by the development of an impaired or altered immune response. Progressive immunosuppression has been observed in patients with acquired immunodeficiency syndrome (AIDS), sepsis, leprosy, cytomegalovirus infections, malaria, cancer and the like. The mechanisms responsible for the down-regulation of the immune response, however, remain to be elucidated.
Deficits in T cell function have been proposed to play an important role in the immune impairment seen in cancer patients and tumor-bearing mice. Mizoguchi (1992) describe alterations in the signal transduction molecules in T cells from MCA-38 tumor-bearing mice that indicate these changes represent the molecular basis for functional impairments observed in splenic T cells isolated from these animals.
An imbalance in the immune system is evident in autoimmunity which is characterized by the production of autoantibodies and autoreactive T cells. The auto-immune disease may be organ-specific in the case of thyrotoxicosis or pernicious anaemia, or non-organ-specific in the case of scleroderma, systemic lupus erythematosus or rheumatoid arthritis. Other diseases which result from the establishment of an autoimmune response include lupus and autoimmune thyroiditis.
On the other hand, hypersensitivity occurs when an immune response occurs in an exaggerated or inappropriate form causing tissue damage. Hypersensitivity reactions are no more than a beneficial immune response acting inappropriately, thereby leading to inflammation and tissue damage. Certain types of hypersensitivity reactions are antibody-mediated while others are mediated primarily by T cells and macrophages.
In Type I hypersensitivity an IgE response is directed against innocuous environmental antigens such as pollen or animal dander. The acute inflammatory reaction with symptoms such as asthma or rhinitis is caused by the release of pharmacological mediators by IgE-sensitized mast cells. Antibody-dependent cytotoxic hypersensitivity or Type II hypersensitivity occurs when antibody binds to either self antigen or foreign antigen on cells. Type III hypersensitivity occurs when immune complexes are formed in large quantities or cannot be cleared adequately by the reticulo-endothelial system. Type IV hypersensitivity is most seriously manifested when antigens are trapped in a macrophage and cannot be cleared. T cells are then stimulated to elaborate lymphokines which mediate a range of inflammatory responses.
T cell recognition events apparently lead to signal transduction and appropriate biochemical signals that control cellular responses. The ability of TCR to transduce signals to multiple biochemical cascades is a central event of immune cell activation. The details of this signal transduction pathway, however, are poorly understood. One or more tyrosine (Tyr) kinases likely have an essential role in T cell activation. Klausner (1991). At least two signal transduction pathways are activated upon stimulation of TCR by an antigen or by monoclonal antibodies directed against either CD3 or the .alpha..beta. heterodimer.
Stimulation of TCR activates a tyrosine kinase. Samelson (1986); Patel (1987); Hsi (1989). Phosphorylation of several proteins with tyrosine residues is induced within seconds of TCR stimulation. June (1990). None of the TCR chains possesses intrinsic kinase activity. A member of the Src family of tyrosine kinases designated Fyn, however, coprecipitates with the CD3 complex. Samelson (1990). A T cell specific member of the Src family of tyrosine kinases, Lck, is tightly, but non-covalently, associated with the cytoplasmic domain of either a CD4 or CD8 molecule. The extracellular domains of CD4 and CD8 bind to MHC class II and class I molecules, respectively. Upon binding of TCR to an antigen-MHC complex on a presenting cell, the TCR is believed to be brought into close proximity with either a CD4 or CD8 molecule that is capable of independently binding to an appropriate MHC molecule.
TCR also activates a phosphatidylinositol-specific phospholipase C which leads to hydrolysis of phosphatidylinositol-4,5-bis-phosphate. Weiss (1984); Imboden (1985). This leads to the liberation of two second messengers: 1) inositol-1,4,5-tris-phosphate which is responsible for transient Ca.sup.2+ mobilization; and 2) diacylglycerol which is a potent activator of protein kinase. Berridge (1989).
Another set of proteins that is related to signal transduction is the NF-.kappa.B/rel transcription factors, also known as the Rel-related protein family. Members of the Rel-related protein family all have similar primary amino acid sequences and bind to an array of homologous decanucleotide sequences with varying affinities. The NF-.kappa.B transcription activator is a multiprotein complex. The NF-.kappa.B transcription activator appears to be specialized in the organism to rapidly induce the synthesis of defense and signalling proteins upon exposure of cells to a wide variety of agents including cytokines, double-stranded RNA, T cell mitogens, DNA damaging agents, protein synthesis inhibitors, parasites, viruses and viral transactivators. A common denominator of the agents that activate NF-.kappa.B is that they either signal or represent a threat to cells and the organisms.
NF-.kappa.B is particularly suited to rapidly activate gene expression because (1) it does not require new protein synthesis, (ii) a simple dissociation reaction triggers activation, (iii) NF-.kappa.B actively participates in cytoplasmic-nuclear signalling and (iv) it is a potent transactivator.
NF-.kappa.B is involved in the inducible expression of the T cell growth factor IL-2, as well as the inducible expression of a component of IL-2 high affinity receptor, suggesting that NF-.kappa.B is a growth regulator. There is indeed a good correlation between the proliferative state of T cells and the state of NF-.kappa.B activity.
Three protein subunits, I.sub..kappa. B, p50 and p65 control the biological functions of NF-.kappa.B. Members of the I.sub..kappa. B protein family display multiple homologous amino acid stretches (ankyrin repeats) that specifically interact with NF-.kappa.B/Rel proteins. IA includes a 35-43 kDa subunit which inhibits the DNA-binding of NF-.kappa.B and serves to retain NF-.kappa.B in an inducible form in the cytoplasm of unstimulated cells. Upon stimulation of cells, I.sub..kappa. B dissociates from the inactive complex with p65 and p50. The released p50-p65 complex heterodimer then migrates into the nucleus and trans-activates genes. Constitutive expression of the IL-2 receptor .alpha. gene in hybrids between a T-cell and myeloma cell line depends solely on the presence of the heterodimer. Only p65 appears to bind I.sub..kappa. B. Within cells, I.sub..kappa. B is released by modification of either I.sub..kappa. B, p65 or both.
Rel proteins are capable of recognizing .kappa.B motifs. The I.sub..kappa. B-family and Rel-family therefore comprise related proteins which are known to be involved in cytoplasmic/nuclear signalling. Other information on the NF-.kappa.B transcription activator and its relationship to the rel proteins may be found in Baeuerle (1991).
The present invention addresses limitations in the art for detecting and monitoring the immune status of a mammal as well as identifying appropriate treatment modalities. The present invention provides improved methods for evaluating the status of a patient's immune system. More specifically, the present invention provides improved methods for identifying, monitoring and evaluating the degree of immunosuppression, hyperimmunity or autoimmunity in a patient.
A need exists for effective methods of measuring the progression of immunosuppression so that attempts at augmenting the immune system in an immunosuppressed patient can be effectively timed. A need also exists for a method by which a patient's level of immunosuppression is estimated and used to accurately predict the likelihood of a patient's response to therapy. A need exists for a method to determine how much to suppress the immune response of a patient with autoimmunity. The patient's therapy can then be developed in a systematic fashion. A method is needed by which a clinician can determine whether a patient's T lymphocytes will be capable of activation and, thus, whether autologous adoptive immunotherapy will likely be efficacious. A need also continues to exist for a method of screening for immunosuppressive agents and agents that reverse or inhibit immunosuppression.
There is a need to detect tumors, in particular early in the development of a tumor, so that treatment effectiveness is enhanced. Also, improved methods for staging of cancer would facilitate choice of the most appropriate treatment modalities. There is also a need to test the effectiveness of treatment modalities prior to clinical trials, and as adjuncts to clinical trials.
There is a need for methods for detecting and measuring the degree of hyperimmunity or autoimmunity in the patient. In addition, improved methods for staging of the progression of hyperimmunity or autoimmunity would facilitate choice of the most appropriate treatment modalities as well as monitor the effectiveness of treatment modalities.
There is a need for methods of monitoring and evaluating the immune status of the patient receiving bone marrow or tissue transplants. Methods for monitoring and evaluating the immune status of the graft recipient, prior to the procedure, as well as after receipt of foreign tissue, are needed to effectively determine when immunosuppressive drugs should be administered.
The present invention addresses limitations in the art for evaluating, monitoring and predicting the status of a patient's immune system thereby providing a means to more effectively diagnose and treat patients with an altered immune status.